LOCUS       sequence-295915-        7579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 3C-like proteinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7579)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7579)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4133870
FEATURES             Location/Qualifiers
     source          1..7579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             920..940
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             974..1051
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             1079..1099
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    1548..1669
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1559..1578)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1613..1632
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1784..1830
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1860..1996
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2041..2112
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2338..2926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2418..2437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3426..4635
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4472..4491
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4639..5238
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4639..4658)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5135..5155
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5265..5313
                     /note="synthetic polyadenylation signal"
     CDS             join(5579..5647,5648..6439)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5797..5816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6452..6588
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6733..6804
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6809..6855)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6873..6921
                     /note="synthetic polyadenylation signal"
     enhancer        7002..7305
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7307..7509
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7460..7480
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7578..7579,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatgggt tttagaaaaa tggcattccc atctggtaaa gttgagggtt gtatggtaca
       61 agtaacttgt ggtacaacta cacttaacgg tctttggctt gatgacgtag tttactgtcc
      121 aagacatgtg atctgcacct ctgaagacat gcttaaccct aattatgaag atttactcat
      181 tcgtaagtct aatcataatt tcttggtaca ggctggtaat gttcaactca gggttattgg
      241 acattctatg caaaattgtg tacttaagct taaggttgat acagccaatc ctaagacacc
      301 taagtataag tttgttcgca ttcaaccagg acagactttt tcagtgttag cttgttacaa
      361 tggttcacca tctggtgttt accaatgtgc tatgaggccc aatttcacta ttaagggttc
      421 attccttaat ggttcatgtg gtagtgttgg ttttaacata gattatgact gtgtctcttt
      481 ttgttacatg caccatatgg aattaccaac tggagttcat gctggcacag acttagaagg
      541 taacttttat ggaccttttg ttgacaggca aacagcacaa gcagctggta cggacacaac
      601 tattacagtt aatgttttag cttggttgta cgctgctgtt ataaatggag acaggtggtt
      661 tctcaatcga tttaccacaa ctcttaatga ctttaacctt gtggctatga agtacaatta
      721 tgaacctcta acacaagacc atgttgacat actaggacct ctttctgctc aaactggaat
      781 tgccgtttta gatatgtgtg cttcattaaa agaattactg caaaatggta tgaatggacg
      841 taccatattg ggtagtgctt tattagaaga tgaatttaca ccttttgatg ttgttagaca
      901 atgctcaggt gttactttcg aaaaccttta ctttcagtcg ggacggcgaa tcccggggct
      961 gataaatccc tggaaaagga gatggaagaa gaactttatt gcagtgtctg cggctaaccg
     1021 gttcaaaaag atcagttcaa gcggcgccct agattacgac attcctacga ccgcctccga
     1081 gaatctctat ttccagggtg aatttggctt ggcacaacat gacgaggccg tcgataacaa
     1141 gtttaataaa gaacagcaga acgctttcta tgagatcctt cacttaccaa atctgaacga
     1201 agagcaacgc aacgctttca ttcagagcct gaaagacgat ccctcgcagt ctgcaaatct
     1261 cctggccgaa gccaagaagc tgaatgacgc acaggctcca aaagttgata acaagtttaa
     1321 caaggagcag cagaatgctt tctacgagat attgcacctt cctaatctga atgaggaaca
     1381 aagaaatgca tttatccagt ccctaaaaga cgatccatca cagagtgcca acctgcttgc
     1441 ggaagccaaa aagctcaatg atgcccaggc cccgaaagta gacgcgaacc atcaataagc
     1501 tagcttgact gactgagata cagcgtacct tcagctcaca gacatgataa gatacattga
     1561 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1621 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1681 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     1741 aaacctctac aaatgtggta ttggcccatc tctatcggta tcgtagcata accccttggg
     1801 gcctctaaac gggtcttgag gggttttttg tgcccctcgg gccggattgc tatctaccgg
     1861 cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc
     1921 agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc
     1981 agaaatttat ccttaaggtc gtcagctatc ctgcaggcga tctctcgatt tcgatcaaga
     2041 cattccttta atggtctttt ctggacacca ctaggggtca gaagtagttc atcaaacttt
     2101 cttccctccc taatctcatt ggttaccttg ggctatcgaa acttaattaa ccagtcaagt
     2161 cagctacttg gcgagatcga cttgtctggg tttcgactac gctcagaatt gcgtcagtca
     2221 agttcgatct ggtccttgct attgcacccg ttctccgatt acgagtttca tttaaatcat
     2281 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2341 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2401 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2461 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2521 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2581 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2641 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2701 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2761 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2821 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2881 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2941 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3001 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3061 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcatttaaat ttccgaactc
     3181 tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg atccatcttg caggctacct
     3241 ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc cttggctatt gcttggcagc
     3301 gcctatcgcc aggtattact ccaatcccga atatccgaga tcgggatcac ccgagagaag
     3361 ttcaacctac atcctcaatc ccgatctatc cgagatccga ggaatatcga aatcggggcg
     3421 cgcctggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcg tcacggcgag
     3481 cgctgccacg tcagacgaag ggcgcaggag cgtcctgatc cttccgcccg gacgctcagg
     3541 acagcggccc gctgctcata agactcggcc ttagaacccc agtatcagca gaaggacatt
     3601 ttaggacggg acttgggtga ctctagggca ctggttttct ttccagagag cggaacaggc
     3661 gaggaaaagt agtcccttct cggcgattct gcggagggat ctccgtgggg cggtgaacgc
     3721 cgatgattat ataaggacgc gccgggtgtg gcacagctag ttccgtcgca gccgggattt
     3781 gggtcgcggt tcttgtttgt ggatcgctgt gatcgtcact tggtgagtag cgggctgctg
     3841 ggctggccgg ggctttcgtg gccgccgggc cgctcggtgg gacggaagcg tgtggagaga
     3901 ccgccaaggg ctgtagtctg ggtccgcgag caaggttgcc ctgaactggg ggttgggggg
     3961 agcgcagcaa aatggcggct gttcccgagt cttgaatgga agacgcttgt gaggcgggct
     4021 gtgaggtcgt tgaaacaagg tggggggcat ggtgggcggc aagaacccaa ggtcttgagc
     4081 ccttcgctaa tgcgggaaag ctcttattcg ggtgagatgg gctgggcacc atctggggac
     4141 cctgacgtga agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc
     4201 agttatggcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt
     4261 gtcgtgacgt cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt
     4321 gtgcggtagg cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg
     4381 aatcgacagg cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg
     4441 tcggttttat gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg
     4501 gggttggcga gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg
     4561 tcaatatgta attttcagtg ttagacttgt aaattgtccg ctaaattctg gccgtttttg
     4621 gcttttttgt tagacaacat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     4681 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     4741 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     4801 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     4861 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     4921 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaggg cctcctggcg
     4981 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     5041 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggccgc cgagcgcgcc
     5101 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     5161 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     5221 cgcaagcccg gtgcctaggc tagtatgtaa gcctagtctt agataataaa atcgctatcc
     5281 atcgaagatg gatgtgtgtt ggttttttgt gtgtgtaacg ctaggcgcgc ctggtgtacc
     5341 gagaacgatc ctctcagtgc gagtctcgac gatccatatc gttgcttggc agtcagccag
     5401 tcggaatcca gcttgggacc caggaagtcc aatcgtcaga tattgtactc aagcctggtc
     5461 acggcagcgt accgatctgt ttaaacctag atattgatag tctgatcggt caacgtataa
     5521 tcgagtccta gcttttgcaa acatctatca agagacagga tcagcaggag gctttcgcat
     5581 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5641 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcgcg
     5701 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5761 agaacgcttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5821 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5881 tgagtattca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5941 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgattgg
     6001 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6061 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6121 tgtagcaatg gcaacaacct tgcgtaaact attaactggc gaactactta ctctagcttc
     6181 ccggcaacag ttgatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6241 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6301 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6361 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6421 actgattaag cattggtaac cgattctagg tgcattggcg cagaaaaaaa tgcctgatgc
     6481 gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca
     6541 acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaaga tcgtttaaac
     6601 tcgactctgg ctctatcgaa tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc
     6661 atttgctcgt cgggcatcga atctcgtcag ctatcgtcag cttacctttt tggcagcgat
     6721 cgcggctccc gacatcttgg accattagct ccacaggtat cttcttccct ctagtggtca
     6781 taacagcagc ttcagctacc tctcaattca aaaaacccct caagacccgt ttagaggccc
     6841 caaggggtta tgctatcaat cgttgcgtta cacacacaaa aaaccaacac acatccatct
     6901 tcgatggata gcgattttat tatctaactg ctgatcgagt gtagccagat ctagtaatca
     6961 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     7021 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     7081 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     7141 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     7201 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     7261 cctacttggc agtacatcta cgtattagtc atcgctatta ccatgctgat gcggttttgg
     7321 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     7381 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     7441 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     7501 agcagagctg gtttagtgaa ccgtcagatc agatctttgt cgatcctacc atccactcga
     7561 cacacccgcc agcggccgc
//
